This invention relates to an apparatus and method for separating formed constituents from the liquid constituent in a biologic fluid sample, so as to facilitate use of an optical instrument to examine the formed constituents. More particularly, this invention relates to an apparatus and method which results in the formed constituents being disposed on a planar surface in the apparatus which planar surface conforms to the focal plane of the optical instrument.
Formed constituents in complex biologic fluid samples are typically isolated in, or separated from, the liquid constituent of the sample so as to enable detailed examination of the formed constituents. Flow cytometry is one technique for identifying formed constituents, such as blood cells in a blood sample. Using this technique, various types of blood cells and other formed constituents in blood can be differentiated from each other, and can be counted. In performing this procedure, the blood sample must be diluted prior to being passed through the flow cytometer. The aforesaid flow cytometry technique does not enable cells or other formed constituents in a blood sample to be quiescently examined. This technique cannot be efficiently used to detect rare events in a blood sample unless the sample is subject to enrichment procedures such as magnetic particle bead enrichment procedures of the type offered by Dynel of Norway.
A second technique for identifying and counting white blood cells and platelets in an anticoagulated whole blood sample is described in U.S. Pat. No. 4,027,660, and in other patents to Robert A. Levine and/or Stephen C. Wardlaw. This technique utilizes a capillary tube having an elongated insert disposed therein. The blood sample is admixed with a stain such as acridine orange, and centrifuged in the capillary tube. The white cells and platelets settle out in the tube between the float and the tube wall so that the white blood cell and platelet layers are elongated by a factor of about ten. The elongation of the cell and platelet layers allows one to ascertain differential white cell and platelet counts in the tube by measuring the distance between opposite cell layer interfaces, and converting the measurements to cell counts. This second technique also does not enable the examination of individual blood cells in the blood sample.
U.S. Pat. No. 6,197,523 describes a method for analyzing a sample of anticoagulated whole blood for the presence or absence of abnormal epithelial cells and/or hematologic progenitor cells. The method involves placing the whole blood sample in a transparent sample tube which includes an insert that occupies sufficient volume in the sample tube so as to form a well defined annular area in the sample tube between the insert and the tube wherein individual cells will be isolated and can be examined. The well defined area of the sample tube is examined under magnification of at least 100X whereby individual cell morphology can be examined therein. As noted, the aforesaid method requires centrifugation of the whole blood sample in the sample tube before the isolated cell can be examined.
A multi-constituent fluid sample can be centrifuged so as to separate the liquid component of the sample from the formed constituents in the sample. This technique is most commonly used for urinalysis. In a biologic fluid sample containing cells or other particulates, the particulates will gravimetrically settle out separately from the liquid constituent, and the cells and particulates will also separate from each other according to their specific gravity. After the sample has been centrifuged, the liquid and the formed constituent fractions of the sample are separated from each other, and one or the other is further analyzed.
In the case of a urinalysis, upon completion of centrifugation, 90 to 95% of the supernatant liquid is decanted or discarded, and the cells and particulates are re-suspended in a smaller amount of the remaining liquid and placed in a chamber, or on a microscope slide for examination. It will be appreciated that the examination of various types of cells or particulates using the centrifugation technique is time-consuming and requires considerable skill on the part of the technician. This technique is also not precise due to the loss or destruction of sample components during centrifugation, and in the case of urinalysis, the imprecision of the decantation and re-suspension steps.
Formed constituents can also be separated from a biologic fluid sample by filtering. Using this technique, the fluid sample is forced to flow through a filter having a pore size which will prevent certain size formed constituents from passing therethrough. Thus if the size of a target formed constituent found in the sample is known, an appropriate filter can be selected for separating that target constituent from the sample. Once the formed constituents are trapped on the filter they can be removed and cultured, or further analyzed. Problems encountered with this technique include the cost of the various filters; the need to know the size of the target formed constituents; the plumbing required to force the sample to flow through the filter; and the potential of filter clogging.
Formed constituents that may be isolated from solutions by centrifugation and/or filtering include: microbes in biologic fluids; casts in urine; somatic cells and blood cells in body fluids, other than blood; cysts; cells from cytological specimens obtained by brushing, aspiration, or scraping, which have been placed in a liquid medium; ova and parasites found In stool samples; and cancerous epithelial and hematologic progenitor cells from anticoagulated whole blood.
As noted above, known techniques for separating formed constituents from a liquid constituent in a biologic fluid sample all include centrifugation of the sample or filtering of the sample. It would be desirable to provide a technique for separating relatively rare formed constituents from a liquid constituent in a quiescent biologic fluid sample, which technique operates in a quiescent manner, is inexpensive, does not require the use of expensive adjunct paraphernalia such as centrifuges, fluid plumbing and filters, and does not require a high degree of expertise and experience to use.
This invention relates to a method and apparatus for use in separating formed constituents from a liquid constituent in an aqueous fluid sample mixture. Candidate aqueous sample mixtures include: urine; cerebrospinal fluid; pleural fluid-, ascites; fluids aspirated from cysts, such as thyroid and breast cysts; cytologic specimens which have been placed in a fluid; aqueous suspensions of stool samples; platelet-rich plasma samples, prepared aqueous bacterial growth mixtures, and the like.
The apparatus of this invention includes a sample chamber which has a transparent sample-viewing portion, and which includes a planar expandable wall, capable of expanding in the presence of aqueous media; such wall being a hydrogel when expanded by addition of aqueous solutions in accordance with the instant invention. The medium, before expansion, may contain an amount of aqueous solution which is below the medium""s equilibrium capacity thus rendering the medium capable of further hydration and expansion, or the medium may be a xero gel, i.,e., a dry polymeric structure that, upon absorption of water, becomes a hydrogel, swelling in the process. For the sake of simplicity, the term xe2x80x9chydrogelxe2x80x9d as used in this application is intended to include any structured water swellable polymeric matrix from the dry state to the fully swollen equilibrium state. The hydrogel-layered wall is disposed opposite to the sample-viewing portion. The layer of the hydrogel has a constant thickness so that the surface of the hydrogel layer which is most proximal to the sample-viewing portion of the chamber is planar. When the apparatus is used to analyze a sample, the chamber is filled with an amount of the sample being examined, the sample being deposited on top of the hydrogel layer. The sample chamber has a known volume, and volume of the layer of the water-absorbant hydrogel is such that, when further hydrated, it will absorb essentially all of the water in the sample and substantially fill the sample chamber with the hydrogel.
After the fluid sample is added to the chamber, the hydrogel will expand toward the sample-viewing portion of the chamber until the chamber is substantially completely filled with the expanded hydrogel. The surface of the expanded hydrogel which is most proximal to the sample-viewing portion of the chamber will remain planar as the hydrogel layer swells. The aqueous constituent of the biologic sample will be absorbed into the hydrogel thereby causing expansion or swelling of the hydrogel. As the hydrogel expands, any formed constituents which are contained in the sample will be captured on the moving planar surface of the hydrogel and will remain in place on that surface of the hydrogel as the hydrogel continues to expand. When the hydrogel has reached its final expanded volume, substantially all of the liquid constituent in the sample will have been absorbed into the hydrogel and all of the formed constituents in the sample will have been captured on the surface of the hydrogel. Since the capture surface of the hydrogel remains planar, and is preferably pressed against the sample-viewing portion of the chamber, the captured formed constituents in the sample will be fixed on a planar surface which can be made to occupy the focal plane of an instrument that is used to examine the captured formed constituents. The various formed constituents which are in the sample and which are captured on the surface of the rehydrated hydrogel can be differentially highlighted by analyte-specific agents so that various formed constituents can be differentiated from each other. Formed constituents can also be stained so that they can be morphologically examined on the hydrogel surface. Various differentially highlighted formed constituents can also be counted. Since the volume of sample added to the chamber is known, formed constituent counts per unit volume of sample can be derived. Isolation and concentration of formed constituents on the hydrogel surface also allows harvesting of specific formed constituents from the hydrogel surface for further analysis of the harvested constituents. The apparatus of this invention can be scanned by an optical scanning instrument, such as a microscope, or an optically differentiating instrument.
It is therefore an object of this invention to provide a method and apparatus for use in obtaining information relating to formed constituents contained in a quiescent biological fluid sample.
It is an additional object of this invention to provide a method and apparatus of the character described wherein the formed constituents in the sample are captured on the planar surface of an expanded hydrogel disposed in a sample container.
It is another object of this invention to provide a method and apparatus of the character described wherein the liquid constituent. In the sample is operative to expand a layer of a water-absorbant hydrogel disposed in the sample container.
It is a further object of this invention to provide a method and apparatus of the character described wherein the planar surface of the expanded hydrogel forms a focal plane for an optical examining instrument which is used in conjunction with the sample container.
It is yet another object of this invention to provide a method and apparatus of the character described wherein individual ones of the formed constituents can be harvested from the planar surface of the hydrogel after the latter has been expanded in the chamber.
It is a further object of this invention to provide a method and apparatus of the character described wherein the number and type of target formed constituents per unit volume of sample can be derived.